PC-AssaySubmit ::= {
assay descr {
aid {
id 652254,
version 1
},
aid-source db {
name "NCGC",
source-id str "USP2605",
date std {
year 2014,
month 1,
day 16
}
},
name "qHTS for Inhibitors of Ubiquitin-specific Protease USP2a Using CHOP2
as the Reporter: Clonogenic Assay",
description {
"Homeostasis of cellular proteins is maintained through a combination of
synthesis and degradation. The pathway that accounts for the majority of
protein degradation is the ubiquitin-proteasomal pathway. Ubiquitin (Ub) is
highly conserved in all cells and the generation of a multi-Ub chain
typically targets proteins for degradation by the proteasome. However,
ubiquitination is highly reversible and dynamic. Deubiquitination, the
reverse process, is catalyzed through the action of enzymes referred to as
isopeptidases or deubiquitinating enzymes (DUBs) [1, 2]. This group of
enzymes is collectively responsible for maintaining adequate pools of free
ubiquitin and regulating the ubiquitination status of cellular proteins. The
class of DUBs referred to as the ubiquitin-specific proteases (USP) family
functions endoproteolytically to cleave Ub chains from a wide range of
protein substrates. USP2a deubiquitinates fatty acid synthase (FASN) which
has recently been identified as an emerging oncology target. To identify
inhibitors of USP2a a cell-free qHTS assay was employed.",
"",
"To further visualize and quantitate the effect of compounds on cell
proliferation, a colony forming or clonogenic assay was performed in 6-well
plates using HCT116 cells. The cells were allowed to adhere to the plates
for 2 days and then compound was added. The cells were allowed to grow for 10
additional days at which point the cells were fixed and labeled with crystal
violet. The colonies were visualized on the Typhoon imager (GE) using Cy5
detection, and quantitated with the associated software. An inhibitor of
USP2 would be expected to lead to a decrease in the size and perhaps the
number of colonies. The cells were cultured in DMEM with 10% FBS and 1%
Pen-Strep and plated in 6-well clear tissue culture treated plates. ",
"NIH Molecular Libraries Probe Centers Network [MLPCN]",
"NIH Chemical Genomics Center [NCGC]",
"",
"MLSCN Grant: MH079852",
"PI Name: Benjamin Nicholson, Progenra Inc, Malvern, PA"
},
protocol {
"2 mL 300 cells/well were dispensed into a 6-well plate in DMEM with 10%
FBS and 1% Pen-Strep and then incubated for 48 hours at 37 deg C, 5% CO2.
Then 3 uL of compound in DMSO was added to each well, which was followed by a
10 day incubation at 37 deg C, 5 %CO2. All medium is then aspirated with a
pipet, the cells are washed with PBS, and then 1 mL of crystal violet in 10%
methanol is added and kept for 30 min at RT. The reagent is then aspirated
with a pipet before the plate is repeatedly submeged in water for washing
puproses. The plate is dried overnight and then read on a GE Typhoon FLA 9500
in end-point mode (Ex 635, Em Long-Pass Red Filter)"
},
comment {
"If a compound has an IC50 <=5 uM, the compound is considered ""active""
and a score of 90 is assigned; if a compound has an IC50 > 5 uM and < 10 uM
then the compound is considered ""inconclusive"" and a score of 50 is
assigned; if a compound has an IC50 >= 10 uM it is considered ""inactive""
and a score of 10 is assigned."
},
xref {
{
xref aid 2281,
comment "Summary AID"
},
{
xref gene 9099
},
{
xref protein-gi 188528692
}
},
results {
{
tid 1,
name "IC50 (uM)",
type float,
unit um
},
{
tid 2,
name "Compound QC",
type string
}
},
revision 2,
activity-outcome-method other,
grant-number {
"MH079852"
},
project-category mlpcn
},
data {
{
sid 162009865,
version 1,
outcome active,
rank 90,
data {
{
tid 1,
value fval { 36, 10, -1 }
},
{
tid 2,
value sval "NCGC"
}
},
date std {
year 2013,
month 4,
day 2
}
}
}
}